Colorectal cancer (CRC) is a potentially preventable disease; however, it still ranks as the third most common cancer and second leading cause of cancer-related deaths in the U.S., with an estimated 50,000 deaths annually. Early detection of clinically relevant colorectal neoplasia (CRN), i.e. CRC and advanced CRAs (A-CRA), is the best way to improve survival. However, available screening modalities to diagnose these lesions are impractical due to invasiveness and cost (i.e., colonoscopy) or insufficient diagnostic accuracy (i.e., fecal-based blood tests). A better approach to screening and surveillance, preferably through noninvasive biomarkers that can facilitate earlier diagnosis of CRC would be highly desirable. MicroRNAs (miRNAs), small non-coding RNAs involved in gene regulation and cancer development, are more robust and stable than larger RNAs, being resistant to degradation in tissues, blood, stool, and other body fluids. Previous studies have achieved limited success in developing definitive sets of miRNA biomarkers for CRC screening due non-comprehensive approaches, and a failure to include all CRNs as meaningful targets for clinical discovery. Also, cohorts used in serum-based studies have been insufficiently powered and lacked independent validation sets. In this proposal, innovative strategies that include Next Generation Sequencing (NGS)-based miRNA-Seq will be applied to permit genome-wide miRNA mapping using tissues and matching sera from patients with CRN, and compared with tissues and sera from individuals without CRN. A novel and powerful new approach is being proposed to identify novel miRNA biomarkers with the highest sensitivity and specificity, which will be validated using large, well-characterized sample sets through the following Specific Aims. Aim 1: Candidate miRNA biomarkers will be discovered using genome-wide NGS approaches in matched tissue and serum specimens from patients with CRN and individuals with a normal colon. Aim 2: Candidate non-invasive miRNA biomarkers will be developed that distinguish patients with CRN vs. those without CRN, and validated using quantitative PCR assays. Aim 3: Clinical validation of prioritized non-invasive miRNA biomarkers identified in Aim 2b will be performed in asymptomatic average risk individuals. The innovation of this project is based upon the first use of a novel, NGS-based miRNA-Seq platform for the biomarker discovery of genome-wide profiling of all miRNAs and iso-miRNAs that are linked to colorectal neoplasia, and validating these using a large, well-characterized cohort of patients with CRN and controls. The long-term goal and potential impact of this project is to develop a sensitive, specific, non-invasive and inexpensive diagnostic test for early colorectal neoplasia.